Method for purifying and enriching molecules

ABSTRACT

The invention relates to a process for purifying and concentrating charge-bearing first molecules  9 , such as proteins, nucleic acids and the like, comprising the following steps: 
     a) preparation of a solution containing the first molecules  9,    
     b) contacting the solution with an electrode  2  which is directly provided with a coating of second molecules  4  having affinity for the first molecules  9 , and 
     c) connecting the electrode  2  to a means  11  for generating an electric field to bring about a movement of the first molecules  9  in the solution directed relative to the electrode  2.

BACKGROUND OF THE INVENTION

The invention relates to a process and an apparatus for purifying andconcentrating charge-bearing first molecules such as proteins, nucleicacids and the like.

Molecules of biological relevance, such as nucleic acids, can beconcentrated for carrying out analytical tests, for example thepolymerase chain reaction (PCR), by precipitating the nucleic acids, bybinding the nucleic acids to a suitable matrix, for example using an ionexchange column, and by various centrifugation methods. Specific nucleicacids can be selected from a nucleic acid mixture by concentrating andseparating in a gel, by hybridizing to membranes or by complexing withspecific proteins. Similar processes are used to purify proteins; alsoused in this instance are processes like high pressure liquidchromatography (HPLC) and antibody-dependent purification processes.Antibody-dependent processes use molecules immobilized on surfaces, suchas, for example, latex beads. Known processes have the disadvantage ofinadequate sensitivity and speed. In addition, they are costly to carryout.

The present invention is based on the object of providing a purificationand concentration process and a corresponding apparatus with which thedisadvantages of the prior art are eliminated. It is additionallyintended to be possible to concentrate in particular nucleic acids,proteins and other charge-bearing molecules of predetermined degree ofhomology or predetermined binding affinity from a large volume.

SUMMARY OF THE INVENTION

According to the process of the invention, the object is achieved by thefollowing steps:

a) preparation of a solution containing the first molecules,

b) contacting the solution with at least one electrode which is directlyprovided with a coating of second molecules having affinity for thefirst molecules, and

c) connecting the electrode to a means for generating an electric fieldto bring about a movement of the first molecules in the solutiondirected relative to the coating.

The electrode is expediently produced from an electrically conductingplastic. It can be a layer on an electrically nonconducting plasticsupport rod or a section, preferably terminal, of such a plastic supportrod. It is also possible for the plastic support rod to be producedcompletely from electrically conducting plastic and to be provided witha handle element which is produced from an electrically nonconductingplastic and can be, for example, slipped on.

On exposure to the electric field there is utilization of the effectthat the first molecules present in solution, for example nucleic acidmolecules, are charge-bearing and thus able to move in the electricfield. The second molecules, which due to exposure to the electric fieldhave come into contact with or reach the direct vicinity of the coating,can bind to the first molecules thereon. Suitable binding in this casemay be, in particular, ionic, covalent, hydrogen bonding or bindingbrought about by steric effects. No electric field may be applied whilethis binding is developing. The process according to the invention canbe used not only when the first molecules are present in a solution. Itis sufficient for the first molecules to be present in a matrix, forexample gel, meat or the like, which permits migration thereof in theelectric field.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWING

Examples of the embodiments of the invention are explained by means ofthe drawing below wherein:

FIG. 1 is a diagrammatic cross-section through a first example of anelectrode embodiment;

FIG. 2 is a diagrammatic cross-section through a second example of anelectrode embodiment;

FIG. 3 is a perspective view according to FIG. 1;

FIG. 4 is a perspective view of a container with an electrode and acounter electrode;

FIG. 5 is a perspective view of a third example of an electrodeembodiment.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

In one embodiment of the invention, the electric field is generated byapplying a first voltage to the electrode so that it acts as anode towhich first molecules bearing a negative charge, for example nucleicacid molecules, are attracted so that binding to the second molecules isachieved. In an alternative embodiment, the electric field is generatedby applying a first voltage to the electrode so that it acts as cathodeto which first molecules bearing a positive charge, for exampleproteins, are attracted so that binding to the second molecules isachieved.

It is regarded as particularly advantageous for the following step to becarried out in particular after the binding of the first to the secondmolecules:

d₁) reversing the polarity and applying a second voltage so that theelectrode acts as cathode from which first molecules bearing a negativecharge, for example nucleic acids, are repelled.

As an alternative to this, the following step can be carried out inparticular after the binding of the first to the second molecules:

d₂) reversing the polarity and applying a second voltage so that theelectrode acts as anode from which first molecules bearing a positivecharge, for example proteins, are repelled.

Blockage of the coating can be prevented by steps d₁) and d₂). This isbecause it is possible, by suitable choice of the second voltage, torepel charge-bearing species which are not bound to the coating from thecoating and thus improve access for further first molecules. It isadditionally possible, by suitably increasing the second voltage, forparticular charge-bearing first molecules to be specifically removedfrom the coating. It is also possible in this way to achieve selectionof particular first molecules. This phenomenon is known as stringency ofa hybridization reaction for nucleic acids.

Care must be taken in the reversal of polarity that the migration ofthose first molecules which have entered into interaction with thesecond molecules or hybridize with the latter is limited. The extent ofthis hindrance depends on the nature and number of the interactions,that is primarily on the degree of homology of the first and secondmolecules interacting with one another, or their affinity. Firstmolecules which have high affinity or complementarity with the secondmolecules are held back most strongly in this case.

It is expedient to carry out the following step during and/or after stepc:

e) heating the electrode or the solution so that the first molecules arethermally dissociated or denatured into their components or subunits,for example single-stranded nucleic acids.

This embodiment makes it possible, for example, to melt double-strandednucleic acids attracted to the coating and thus facilitates the bindingof the single strands to, for example, complementary oligonucleotidesprovided in the coating. Keeping the surface at a particular temperaturethus also makes a contribution to the stringency of the selection ofparticular first binding molecules.

It is expedient to carry out the following step in particular after stepd₁ or d₂:

f) cooling the electrode or the solution to bring about binding of thefirst molecules, components and/or subunits thereof to the secondmolecules.

Step f additionally assists the binding of the first molecules, theircomponents or subunits to the coating.

It is possible, depending on the nature of the first molecules, torepeat one or more of steps c-e.

It is expedient, especially during and/or after step d₁ or d₂, to mixthe solution, preferably mechanically. Removal of first molecules,components and/or subunits repelled from the electrode by steps d₁ andd₂, is assisted in this way.

The maximum values of the first and second voltage are advantageouslychosen so that degradation of the first and second molecules, and thecomponents and/or subunits, by electrolysis is avoided. It hasadditionally proven beneficial to choose the maximum value of the secondvoltage so that breaking of linkages formed between the first moleculesor their components or subunits and the second molecules is avoided.

It is possible, by adapting to the nature of the first molecule to beconcentrated or purified, to control, preferably automatically, themaximum values of the first and/or second voltage(s), the duration ofthe polarity and reverse polarity and/or the temperature of theelectrode as a function of parameters of the solution such as its pH,ionic conductivity, concentration of first molecules, temperature andthe like.

According to the achievement in terms of apparatus, an apparatus forconcentrating charge-bearing first molecules present in a solution, suchas nucleic acids, proteins and the like, is provided with an electrodewhich is directly provided with a coating of second molecules havingaffinity for the first molecules, and where the electrode is connectedto a means for generating an electric field so that it is possible tobring about a movement of the first molecules in the solution directedrelative to the electrode.

This means that an apparatus for carrying out the process according tothe innovation is made available with which the disadvantageous blockingof the coating is avoided and it is possible to bring aboutconcentration of specific molecules on a surface. The electrode can beproduced simply and cheaply; it can therefore be used as disposablearticle.

The electrode is preferably produced from an electrically conductingplastic, in particular a polycarbonate, a polycarbene, polycarbonatecopolymer or homopolymer with a conducting additive such as graphite.Electrically conducting plastics of this type are disclosed, for examplein DE 35 41 721 A1, the contents of which are incorporated herein byreference.

The means for generating the electric field can be a means forgenerating an alternating electric field. It is thus possible to achieverapid and specific occupation of the coating with the first molecules tobe concentrated or purified.

Depending on whether the first molecule has a positive or negativecharge, the means for generating the electric field may comprise theelectrode as anode or as cathode.

The plastics present as electrode material permit the chemistry ofcoating (Hitoshi Kohsaka: J. Clin. Lab. Anal. 8:452-455 (1994)) with thesecond molecules predetermining the selection criteria to be very simpleand thus low-cost. The coating provided on the electrode has, accordingto another embodiment feature, at least one aliphatic radical which ispreferably linked to the electrode via an NH or SH linkage. Thealiphatic radical may in this case have a chain length of 2-20,preferably of 6-10, carbon atoms. A protein sequence, peptide sequenceor nucleotide sequence is expediently linked to the free end of thealiphatic radical. The nucleotide sequence may comprise 5-30nucleotides. It is moreover advantageous for the nucleotide sequence tobe a first oligonucleotide formed by a chain of 10-20, preferably 15,nucleotides. It is regarded as particularly advantageous to link asecond oligonucleotide, preferably via a sugar-phosphate linkage, to thefirst oligonucleotide.

The electrode can be provided with a heating element which is separatedfrom the electrode by an electrical insulator. The insulator can beproduced from glass or ceramic, preferably from aluminum oxide ornitride. The heating element can be a resistance heating elementproduced from platinum. The resistance heating element can, in oneembodiment of the invention, also be identical to the electrode because,with suitable circuitry, the high electrical resistance, by comparisonwith the leads, of the plastic electrode can be used to heat theelectrode surface.

In order to divert unwanted molecules away from the coating and bringabout redistribution of first molecules to be purified and concentratedin the solution, it is expedient to provide a means for mixing thesolution. This may be a stirrer which can be operated electrically or astream of gas passed through.

According to another embodiment feature of the invention, the electricalquantities for generating the electric field and for operating theheating element and the means for mixing can be controlled, preferablyautomatically, as a function of parameters of the solution such as itspH, ionic conductivity, concentration of first molecules, temperatureand the like. A computer will expediently be used for automatic control.

Finally, a combination of means for producing the apparatus according tothe innovation and for carrying out the process according to theinnovation are claimed.

EXAMPLE 1 Construction of the Coating and of the Element LocatedUnderneath

A first insulating layer which can be formed from glass or ceramic, forexample from aluminum oxide, is applied to a support rod. To this isapplied a conducting layer, for example produced in the form of aplatinum zigzag, for heating. The conducting layer is in turn covered bya thin second insulating layer. It may have a thickness of 150 μm and beproduced from glass. On this is located an electrode formed from gold.It is bonded.

The coating is provided on the electrode. C6 aliphatic linker moleculesare linked via SH groups to the surface of the electrode. Spacermolecules, for example oligonucleotides consisting of 10 thymidineresidues, are in each case linked to the free end of the linkermolecules. 10 pmol of a 20-mer with a predetermined sequence is linkedin each case to their free ends via a sugar-phosphate linkage. Theelectrode provided with the coating is normally used in combination withthe claimed apparatus. However, it may also relate to a separateinnovation.

EXAMPLE 2 Purification

The electrode described in Example 1 is immersed in 1 ml of ameasurement solution containing 20 pmol of radiolabeled oligonucleotide(20-mer) and 40 pmol of DNA single strands. The oligonucleotide iscomplementary to the oligonucleotide bound in the coating, whereas theDNA single strands are not. The following results are obtained for theactivity of the coating determined by means of Cherenkov counting:

Application of a voltage of 0.2 V and a current of 0.8 mA to theelectrode results, after reversal of the polarity several times, in abinding of 9% of the total activity to the coating. On-connection of theelectrode as anode for two minutes without reversal of polarity there is2% binding of the total activity. Without application of a voltage, lessthan 0.2% of the total activity is bound to the coating.

A support rod 1 produced from Teflon is provided with a conducting layeror electrode 2 consisting of an electrically conducting plastic. Thesurface 3, facing the solution (not depicted here), of the electrode 2is coated with oligonucleotides 4. A first lead 5 is embedded in thesupport rod 1 and is bonded to the electrode 2. Two other second leads 6(depicted here by a broken line) can likewise be bonded to the electrode2 for heating.

In the example of an embodiment shown in FIG. 2, an electricallyinsulating intermediate layer 7 is additionally provided between theelectrode 2 and the support rod 1. It separates the electrode 2 from aheating layer 8 which is provided directly on the support rod 1 producedfrom Teflon.

FIG. 3 shows a perspective view of the example of an embodimentdescribed in FIG. 1. 9 designates chargebearing first molecules presentin a solution.

FIG. 4 shows the example of an embodiment according to FIG. 3 inperspective view. The support rod 1 is immersed in a solution which isaccommodated in a container 10 and in which first molecules 9 arepresent. The first lead 5 is connected to a source of alternatingvoltage 11. Likewise connected to the source of alternating voltage 11is, via a third lead 12, a counter electrode 13 immersed in thesolution.

FIG. 5 shows a perspective view of a third example of an electrodeembodiment. The surface 3 of electrode 2 is present at the tip of thesupport rod 1 produced from insulating plastic. The embedded first lead5 is connected to a first connecting socket 14 and the second leads 6are each connected to second connecting sockets 15. Plugs of connectingcables can be inserted into connecting sockets 14 and 15.

The apparatus functions in the following way:

In order to isolate biomolecule samples from a solution, an apparatusaccording to the invention is immersed in the solution. Then a voltageis applied through the electrode 2 provided on the support rod 1 andthrough a counter electrode 13 immersed in the solution. Depending onthe polarization of electrode 2, this brings about electrophoreticmigration of oppositely charged biomolecules in the direction ofelectrode 2. When the coating consisting of oligonucleotides 4 isreached, the latter are bound thereto.

In the case of double-stranded DNA, the polarization of electrode 2 isinterrupted after a predetermined time. Heating of electrode 2 thentakes place. This breaks down the DNA double strands adhering tooligonucleotides 4 into their components. Reapplication of thepolarization moves the single strands back to the oligonucleotides 4,where they are bound. Samples can be obtained simply and rapidly fromthe solution in this way.

List of Reference Numbers

1 Support rod

2 Electrode

3 Surface

4 oligonucleotides

5 First lead

6 Second lead

7 Intermediate layer

8 Heating layer

9 First molecules

10 Container

11 Source of alternating voltage

12 Third lead

13 Counter electrode

14 First connecting socket

15 Second connecting socket

What is claimed is:
 1. A process for concentrating chargebearing firstmolecules from a crude solution, comprising the following steps: a)preparing a crude solution containing the first molecules, b) contactingthe solution with an electrode which is directly provided with a coatingof second molecules having affinity for the first molecules, saidelectrode consisting of conducting plastic, c) connecting the electrodeto a means for generating an electric field, d) generating an electricfield effective to direct the movement of the first molecules relativeto the electrode, e) binding a portion of the first molecules to aportion of the second molecules, f) reversing the polarity of theelectrode to repel chargebearing species that are not bound to thesecond molecules to improve access for further first molecules, and g)concentrating said first molecules by way of steps a) through f).
 2. Theprocess as claimed in claim 1, wherein to generate the electric field afirst voltage is applied to the electrode so that it acts as an anode towhich the first molecules bearing a negative charge are attracted sothat binding to the second molecules is achieved.
 3. The process asclaimed in claim 2, wherein the following step is carried out after thebinding of the first molecules to the second molecules: after the stepof generating the electric field, reversing polarity of the field andapplying a second voltage so that the electrode acts as a cathode fromwhich first molecules bearing a negative charge are repelled.
 4. Theprocess as claimed in claim 3, wherein the following step is carried outduring step c: heating the electrode or the solution so that the firstmolecules are thermally dissociated or denatured into their componentsor subunits.
 5. The process as claimed in claim 4, wherein the followingstep is carried out after the step of reversing the polarity: coolingthe electrode or the solution to bring about binding at least one of thefirst molecules, components or subunits to the second molecules.
 6. Theprocess as claimed in claim 5, wherein one or more of the recited stepsare repeated.
 7. The process as claimed in claim 6, wherein the solutionis mixed.
 8. The process as claimed in claim 3, wherein maximum valuesof the first and second voltages are chosen so that degradation ordissociation of the first and second molecules, and the components andsubunits, by electrolysis is avoided.
 9. The process as claimed in claim3, wherein a maximum value of the second voltage is chosen so thatbreaking of linkages formed between at least one of the first molecules,their components, or their subunits, and the second molecules isavoided.
 10. The process as claimed in claim 3, wherein a maximum valueof at least one of the first voltage, the second voltage, the durationof the polarity and reverse polarity or the temperature of the electrodeare controlled as a function of at least one physical parameter of thesolution.
 11. The process as claimed in claim 1, wherein to generate theelectric field a first voltage is applied to the electrode so that itacts as a cathode to which the first molecules bearing a positive chargeare attracted, so that binding of the first molecules to the secondmolecules is achieved.
 12. The process as claimed in claim 11, whereinthe following step is carried out after the binding of the firstmolecules to the second molecules: reversing the polarity of the fieldand applying a second voltage so that the electrode acts as an anodefrom which first molecules bearing a positive charge are repelled. 13.The process as claimed in claim 12, wherein the following step iscarried out during step c: heating at least one of the electrode and thesolution, so that the first molecules are thermally dissociated ordenatured into their components or subunits.
 14. The process as claimedin claim 13, wherein the following step is carried out after the step ofreversing the polarity of the electric field: cooling at least one ofthe electrode and the solution to bring about binding at least one ofthe first molecules, components or subunits to the second molecules. 15.The process as claimed in claim 14, wherein one or more of the recitedsteps are repeated.
 16. The process as claimed in claim 15, wherein thesolution is mixed.
 17. The process as claimed in claim 16, whereinmaximum values of the first and second voltage are chosen so thatdegradation or dissociation of the first and second molecules, and thecomponents and subunits, by electrolysis is avoided.
 18. The process asclaimed in claim 17, wherein maximum value of the second voltage ischosen so that breaking of linkages formed between the first moleculesor their components or subunits and the second molecules is avoided. 19.The process as claimed in claim 18, wherein a maximum value of at leastone of the first voltage, the second voltage, the duration of thepolarity and reverse polarity or the temperature of the electrode arecontrolled as a function of at least one physical parameter of thesolution.
 20. The process as claimed in claim 3, wherein the firstmolecules are nucleic acids.
 21. The process as claimed in claim 3,wherein the following step is carried out after step c: heating theelectrode or the solution so that the first molecules are thermallydissociated or denatured into their components or subunits.
 22. Theprocess as claimed in claim 4, wherein said components comprisesingle-stranded nucleic acids.
 23. The process as claimed in claim 21,wherein said components comprise single-stranded nucleic acids.
 24. Theprocess as claimed in claim 7, wherein the solution is mechanicallymixed.
 25. The process as claimed in claim 10, wherein the physicalparameter is selected from the group consisting of pH, ionicconductivity, concentration of first molecules and temperature.
 26. Theprocess as claimed in claim 11, wherein the first molecules compriseproteins.
 27. The process as claimed in claim 12, wherein the firstmolecules comprise proteins.
 28. The process as claimed in claim 12,wherein the following step is carried out after step c: heating theelectrode or the solution so that the first molecules are thermallydissociated or denatured into their components or subunits.
 29. Theprocess as claimed in claim 13, wherein said components comprisesingle-stranded nucleic acids.
 30. The process as claimed in claim 28,wherein said components comprise single-stranded nucleic acids.
 31. Theprocess as claimed in claim 16, wherein the solution is mechanicallymixed.
 32. The process as claimed in claim 19, wherein the physicalparameter is selected from the group consisting of pH, ionicconductivity, concentration of first molecules and temperature.
 33. Theprocess as claimed in claim 2 wherein the first molecules are nucleicacids.